Felis Concolor Coryi)

J. Helminthol. Soc. Wash. 64(1), 1997 pp. 137-141

Microfilariae in the Free-Ranging Florida Panther (Felis concolor coryi)

MARNIE G. LAMM,' MELODY E. RoELKE,2-5 ELLIS C. GREiNER,3-6 AND CHRISTINE K. STEiBLE4 1 College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610 (e-mail: [email protected]), 2 Florida Game and Fresh Water Fish Commission, Gainesville, Florida 32610, 3 Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32610 (e-mail: [email protected].), and 4 Department of Statistics, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611

ABSTRACT: Blood samples from Florida panthers (Felis concolor coryi) collected from 1986 to 1993 during the months of December through May were screened for the presence of microfilariae (mff) by the Difil® filter test. Thirty-five of 47 (74.5%) panthers older than 2 yr of age were positive with microfilaremias ranging from 10 to 7,380 mff/ml of whole blood. No panthers that were 6 mo of age or less (n = 10) were microfilariae- positive, and only 20% of the panthers in the 1-yr class (n = 5) were positive. A representative number of microfilariae (n = 40) from each of 7 freshly collected positive blood samples was measured and morphological characteristics were noted. The average length of microfilariae processed by the modified Knott's technique was 320 /Am (273-370 /Am) with a width of 4-5 /Am. Of the 280 microfilariae measured, 202 (72.14%) had tapered heads and straight tails with an average length of 319 /im (276-368 /Am), 61 (21.79%) had blunt heads and straight tails and averaged 323 /urn (274-366 /Am), 16 (5.71%) had tapered heads and button-hooked tails with an average length of 320 /Am (290-368 /am), and 1 (0.35%) had a blunt head and button-hooked tail and measured 320 /Am. The finding of no significant difference (P > 0.05) between length measurements due to differences in head and tail shape leads us to believe that all microfilariae were of 1 species. Based on microfilarial length measurements, review of necropsy reports, and comparison with bobcat microfilariae, the most likely filarial species infecting the Florida panther is Dirofilaria striata (Molin, 1858). KEY WORDS: Florida panther, Felis concolor coryi, Dirofilaria striata, microfilariae, morphology, prevalence, bobcat, Lynx rufus.

Filarial nematodes have been reported in a been found in the ocelot (Felis pardalis), the number of exotic felid species. Dirofilaria re- margay (Felis tigrina) (Anderson and Diaz-Un- pens has been reported in the lion (Panthera leo) gria, 1959), and the bobcat (Orihel and Ash, (Nelson et al., 1962). Dirofilaria immitis has 1964; Miller and Harkema, 1968; Roelke, un- been found in jaguars (Felis onca), tigers (Felis publ. 1985). Forrester et al. (1985) reported sondiacus and Felis tigris), wild cats (Felis adult D. striata in mff+ adult Florida panthers. bangsi costariensis), jagouarundi (Felis yago- The Florida panther is an endangered subspe- uarundi) (Otto, 1974), a bengal tiger (Panthera cies of cougar that inhabits pockets of habitat in tigris) (Kennedy and Patton, 1981), and bobcats the Big Cypress and Everglades ecosystems of (Lynx rufus) from Florida (Levine, 1980; For- southern Florida (U.S.A.). A population of less rester, 1992). Of 22 free-ranging mountain lions than 40 adult animals remains in the wild (Bel- (Felis concolor) from California, 1 was seropos- den, 1986). Since veterinary involvement in the itive for antibodies against the somatic antigen Florida Panther Recovery Program began in of D. immitis yet was seronegative for cuticular 1983, an intensive health evaluation and moni- antigen and no circulating microfilariae (mff) toring program has been in effect. The protocol were found in the whole blood (Paul-Murphy et includes the collection, screening, and storage of al., 1994). Dirofilaria striata was first reported various biological samples each time an animal in Brazilian pumas (Felis concolor and F. ma- is immobilized. In the past, the presence of mff croura) (Raillet and Henry, 1911) and since has has been noted either by buffy coat analysis, by membrane filtration, or in reports from clinical laboratories. Adult D. striata were found in 4 5 Present address: Tanzania National Parks, Arusha, Tanzania, Africa. out of the 7 panthers examined from 1973 to 6 Corresponding author. 1983, 3 of which were mff+ (Forrester et al.,

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1985). It was assumed that all microfilariae from high numbers of mff were chosen for morphological these animals were representative of D. striata study of individual microfilariae. All blood samples were kept in EDTA-coated blood tubes for the 2-8 hr because no other adult filariids were detected. between time of collection and time of microfilarial Adult D. striata were found in 5 additional analysis. A 1-ml aliquot was processed using a modi- microfilaremic panthers at necropsy (Roelke, fied Knott's procedure (Knott, 1939). Microfilariae Nayer, and Vickery, unpub. data 1985). Accord- were measured immediately after processing in order to minimize possible effects on length or width of mi- ing to necropsy reports, 1-3 adult filariids were crofilariae due to storage in 2% formalin. Measure- present singularly in the fascia between muscle ments and morphological assessments were made with bundles in the distal extremities. No reports of a calibrated ocular micrometer at a magnification of any filarial species other than D. striata were X400. Length, head shape, tail shape, and body shape found in review of Florida panther necropsy re- were recorded for 40 mff/sample. Head shape was con- sidered tapered if the width decreased upon successive ports (n = 41) from 1980 to September 1994. measurements anteriorly (Fig. 1); if the sides remained This study was undertaken to determine the parallel, the head was categorized as blunt (Fig. 2). prevalence and microfilaremia of Dirofilaria sp. Although many posterior ends were curved, only those in the Florida panther population and to char- that had a distinct hook at the tip were labeled as such (Fig. 1,4). acterize and measure the mff in order to confirm Data were analyzed using SAS (SAS Institute Inc., the identity of the species of filariid present. 1989). Comparisons of total lengths of the microfilariae were made between panthers and bobcats and be- Materials and Methods tween morphological types using ANOVA with a split- plot design where felid type was the whole-plot factor From 1986 to 1993, a total of 83 blood samples and morphological type was the sub-plot factor. Three representing 47 individual panthers and 3 bobcats was morphological types (tapered head and straight tail, screened for the presence of mff. Samples were col- blunt head and straight tail, and tapered head and but- lected during the field capture season in the months of ton-hooked tail) were included in this analysis. Micro- December through May from free-ranging panthers in filariae with blunt head and button-hooked tails were the Big Cypress and Everglades ecosystems. All ani- not included due to low prevalence (1 found in pan- mals were anesthetized with ketamine hydrochloride thers and 3 found in bobcats). Confidence limits were (Ketastat®, Bristol Laboratories, Syracuse, New York, not calculated for percentage of prevalence of mff in U.S.A.) or a combination of tiletamine/zolazepam (Tel- panthers because the number examined represented azol®, A. H. Robins Co., Richmond, Virginia, U.S.A.). nearly all of the extant population of panthers, thus Blood was drawn prior to administration of fluids, vac- rendering confidence limit calculations moot. cinations, and prophylactic injections of Ivermectin (Ivomec®, Merck & Company, Incorporated, Rahway, New Jersey, U.S.A.) at a dose of 200 meg/kg. Blood Results and Discussion samples were taken directly from the saphenous vein Thirty-five (74.5%) of 47 adult (>2 yr of age) into sterile 3-ml tubes containing ethylenediaminetet- Florida panthers sampled from 1986 to 1993 raacetic acid (EDTA) via a vacutainer and butterfly apparatus. The sample was kept cool in an insulated were mff+ at some point in their lives and all container for the 2-8-hr transition from field to lab. except for 2, which had low counts of 20 and One milliliter of EDTA preserved whole blood from 330 mff/ml, remained positive on subsequent each of the 17 samples collected in 1992 and 5 sam- tests. Because panthers were usually sampled at ples from 1993 was processed using the Difil® (EV- SCO Pharmaceuticals, Buena, New Jersey, U.S.A.) 2-yr intervals, the average age when animals be- procedure described by Howland and Todd (1977). In come mff+ is not precisely known. None of the addition, 58 whole blood samples preserved in EDTA 5 panthers tested at 6 mo of age were mff+, 2 that had been collected from free-ranging panthers of 10 (20%) tested positive in the 1-yr-old class, from 1986 to 1991 and stored in a 1:10 ratio of blood and 15 of 23 (65%) of panthers were test posi- (1 ml) to 2% formalin (9 ml) at 4°C were screened for mff. From the 10-ml sample, 5 ml were passed through tive at 2-4 yr of age. Of the 23 panthers that a Difil filter. Numbers of mff were determined for all were 10 yr of age or older, 22 (96%) were mff+. positive samples (except FP#5 1 in which high numbers Counts ranged from 10 to 7,380 mff/ml of of mff/field made accurate counting impossible) by whole blood. Microfilarial counts showed fluc- counting all individual mff on the filter at a viewing magnification of X200 to obtain a mff/1 ml value. tuations with no apparent trends when compar- When no mff were found, the remaining 5 ml were isons were made between animals or over peri- tested in the same manner to confirm that the entire odic sampling of individual panthers. Adminis- sample was indeed negative. In all cases, the Difil filter tration of ivermectin at the time of capture holder was washed and dried well between samples in seemed to have no effect on the long-term mi- order to avoid cross-contamination leading to false- positive results. crofilarial intensities. Because the panther-cap- Of the 17 samples screened in 1992, 7 samples with ture interval was normally every 2 yr, it was

Figure 1. Microfilaria from panther blood with tapered head and hooked tail. Total length = 332 u.m. Figure 2. Microfilaria from panther blood with blunt head and straight tail. Total length = 319 jxm. Figure 3. Microfilaria from panther blood with tapered head and straight tail. Total length = 329 m. Figure 4. Microfilaria from panther blood with blunt head and hooked tail. Total length = 323 u.m. impossible to assess the short-term effect of this head, straight tail (TS); and tapered head, but- anthelmintic on microfilaremias in the Florida ton-hooked tail (TH). Microfilariae with tapered panther. heads and straight tails were the most prevalent Panther microfilariae were of 4 morphological (72.1%), followed by those with blunt heads and types (Fig. 1-4): blunt head, straight tail (BS); straight tails (21.8%). The next most common blunt head, button-hooked tail (BH); tapered were mff with tapered heads and button-hooked

Copyright © 2011, The Helminthological Society of Washington 140 JOURNAL OF THE HELMINTHOLOGICAL SOCIETY OF WASHINGTON, 64(1), JAN 1997 itu J\.JUIM>/AI^ *ur i no ncLivuiN i rujLAjiju^rtL OUV^IE, i i v^r wrtonn-svj IUIN, otvi;, J/\IN iyy I tails (5.7%) and, lastly, those with blunt heads Table 1. Distribution and average lengths of Flor- and button-hooked tails (0.4%). Body shapes ida panther and Bobcat microfilariae based on morphology. were not used as criteria for distinguishing filarial types because shape appeared to be a highly X Range variable parameter. Head/tail N % SD (M-m) The average lengths of the mff from panthers measured in fresh blood samples (n = 280) was Tapered/straight Panther 202 72.14% 319 7.16 276-368 320 /mi (273-370 /mi). The widths ranged from Bobcat 87 72.5% 333 6.16 297-363 4 to 5 /mi. All of the mff measured remained in their original whole blood sample for a period Blunt/straight Panther 6 1 21.79% 323 8.25 274-366 of 2-8 hr before analysis. Courtney and Garber Bobcat 24 20.0% 326 6.14 299-350 (1983) found, in measurement of D. immitis and D. reconditum mff collected from dogs and Tapered/button-hooked Panther 16 5.71% 320 8.52 290-368 stored in EDTA for 8 hr, that there was not a Bobcat 6 5.0% 338 6.17 311-350 significant change in microfilarial width or length. In an earlier study, Sawyer et al. (1963) Blunt/button-hooked determined that the width of D. reconditum Panther 1 0.35% 320 0 320 Bobcat 3 2.5% 339 7.02 322-354 stored in blood for 4-6 hours increased an average of 0.5 /mi above the average of those measured immediately. Both authors concluded that a more sensitive detection method by House and there was no change in morphological features Glover (1974), who reported that the Difil test during this time. is 97.5% accurate whereas the Knott's procedure Because bobcats and panthers inhabit the is only 89% accurate. same habitat and D. striata has been reported in From review of necropsy data and the mor- the bobcat, a screening of 3 bobcat blood sam- phological data presented here, it can be inferred ples and comparison of mff between the 2 spe- that D. striata is the only species of filariid pres- cies was performed. Of the 3 bobcats examined, ent in the Florida panther. Comparison of the all had high microfilaremias. The average length microfilarial length values with those published of bobcat mff measured (n = 120) was 333 /mi for various Dirofilaria spp. shows that they are (297-363 /mi) and the average width was 5 /mi most similar to D. striata, although references (4-6 /mi). A majority of the mff (72.5%) had regarding measurements of this filariid in felids tapered heads and straight tails, 20% had blunt are few and reported microfilarial lengths vary heads and straight tails, 5% had tapered heads significantly depending on processing methods. and hooked tails, and 2.5% had blunt heads and Anderson and Diaz (1959) isolated unsheathed button-hooked tails. This distribution of mor- mff from the uterus of adults with a length of phologic types is similar to that of the panther 235-270 /mi and a width of 5 /mi. The mea- (see Table 1). Neither felid type, morphological surements of formalin-fixed D. striata from bob- type, nor the interaction between felid type and cats reported by Orihel and Ash (1964) averaged morphological type had a significant effect on 348 /mi (327-371 /mi) long by 4-5 /mi wide, microfilarial length (P = 0.3375, 0.3572, and and those measured from hematoxylin-stained 0.4178, respectively). thick blood films were 230-240 /mi. Redington Knott's procedure was the chosen method for et al. (1977) reported finding similar dimensions studying morphological features because it though no values were given. The finding of caused less deformation and better delineation such a high prevalence of mff in adult Florida of the worms than did the Difil test. Jackson panthers warrants definitive microfilarial identi- (1977) noted that the filtration step of the Difil fication as well as continued screening of whole test procedure seemed to cause shrinkage in blood samples and investigation into the subtle length as compared to those measurements ob- health effects that such high numbers of circu- tained from a Knott's preparation and that the lating mff may pose. filter membrane would often trap the mff mid- way through and obscure observation of the tail. Acknowledgments The Difil method was used to obtain microfilar- The authors thank N. G. Keeling, A. J. An- ial counts because it was faster and was deemed derson, R. Ball, R. T McBride, D. S. Maehr, E.

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